MAGE genes: Prognostic indicators in AL amyloidosis patients

Abstract A high frequency of MAGE‐CT (cancer testis) antigens are expressed in Multiple Myeloma (MM) patients; however, in other plasma cell dyscrasias, their potential function remains unclear. We measured the expression of MAGE‐CT genes (MAGE‐C1/CT7, MAGE‐A3, MAGE‐C2/CT10) in 105 newly diagnosed amyloid light‐chain (AL) amyloidosis patients between June 2013 and January 2018 at Peking University People's Hospital using real‐time quantitative polymerase chain reaction. In the newly diagnosed AL patients, the positive expression rates of patients with MAGE‐C1/CT7, MAGE‐C2/CT10 and MAGE‐A3 were 83.8% (88/105), 56.71% (38/67) and 22.0% (13/59) respectively. There was no significant correlation between organ propensity and MAGE‐CT gene expression. Changes in the MAGE‐C1/CT7 levels were consistent with a therapeutic effect. The expression levels of MAGE‐C1/CT7, MAGE‐C2/CT10 and MAGE‐A3 provide potentially effective clinical indicators for auxiliary diagnoses and monitoring treatment efficacy in AL amyloidosis patients.


| INTRODUC TI ON
Amyloidosis is a disorder that is characterized by misfolded insoluble protein fibrils deposited in a variety of tissues and organs. Amyloid light-chain (AL) systemic amyloidosis is one form of systemic amyloidosis and is caused by plasma cell dyscrasia. Current therapies, including novel drugs and autologous stem cell rescue, significantly improve the survival of these patients. However, some patients are still not responsive to anti-plasma cell therapy. [1][2][3] Therefore, it is necessary to understand the biological characteristics of tumour plasma cells in AL amyloidosis. The mechanisms responsible for tumorigenic plasma cells in AL amyloidosis involve a series of genetic alterations in the bone marrow microenvironment that promote tumour growth and the failure of the immune system to recognize it. 4,5 The cancer-testis families of tumour-associated antigens (CT antigens) were originally discovered in patients with malignant melanoma. These antigens are expressed in a broad range of human tumours, while they are limited to developing germ cells in normal tissue and occasionally the placenta. 6 Additionally, MAGE-CT antigens are able to elicit cytotoxic T cells and humoural responses.
Because CT antigens show restricted normal tissue expression and are highly immunogenic, they are attractive targets for diagnostic value as tumour markers and immunotherapeutic approaches in cancer patients. Regarding Multiple Myeloma (MM), our previous study and other research show that MAGE-CT antigens are expressed with a frequency of 59%-92.3% in symptomatic MM patients. [7][8][9] However, the study of CT antigens is mainly concentrated in myeloma, and there is little research in areas of other plasma cell dyscrasia including AL amyloidosis. 10 To investigate the relationship between MAGE-CT antigen expression and AL amyloidosis, we measured the messenger ribonucleic acid expression of primary bone marrow specimens from newly diagnosed AL amyloidosis patients by real-time polymerase chain reaction (RT-PCR) and analysed their relationship with the prognosis of patients.

| Patients
Between June 2013 and Jan 2018, 105 AL amyloidosis patients who underwent MAGE CT gene detection were enrolled in this study. AL amyloidosis was diagnosed by the presence of Congo Red-positive fibril deposition upon biopsy and evidence of monoclonal protein upon serum protein electrophoresis, serum/urine immunofixation electrophoresis, or free light chain (FLC) analysis.
Immunofluorescence or immunoelectron microscopy was used to identify the AL subtype. Organ involvement was assessed according to the consensus criteria. 11 Baseline data at diagnosis were extracted from medical records, whereas follow-up information was recorded after each visit. CD138-enriched (fluorescence in situ hybridization, FISH) panels for cytogenetics from bone marrow were collected from patients. FISH panels included t(4;14), t(14;16), t (11;14), del17p, +1q21. If the proportion of bone marrow clonal plasma cells ≥10% in one patient without bone destruction, hypercalcinaemia and tubular nephropathy AL amyloidosis was still diagnosed. The definition of plasma cell dyscrasia was established according to the definitions of the international myeloma working group. [12][13][14] Informed consent was obtained from all patients prior to their enrolment in the study. The study design adhered to the principles of the Helsinki Declaration and was approved by the ethics committee of Peking University People's Hospital.

| Real-time quantitative PCR for MAGE-CT antigens
Bone marrow samples were collected from patients with AL amyloidosis during routine diagnostic procedures at the Peking University People's Hospital. The real-time quantitative PCR technique in our laboratory was described in detail previously.

| Treatment response and outcome
A total of 66 out of 105 patients received bortezomib-based chemotherapy. Haematological response and organ response were evaluated according to the consensus guidelines. 15

| Statistical analysis
The χ 2 or Fisher's exact tests were used for categorical variables, whereas a t test or nonparametric test was used for continuous variables. Standard deviation for the positive distribution and quartiles for the non-normal distribution were calculated to compute the standard. End-points were calculated at the time of last contact; the overall survival (OS) was defined from the first time of relapse to the last contact or the time of death. A survival curve was generated using the Kaplan-Meier method. Comparison of survival was performed by the log-rank test. A P < 0.05 denoted statistical significance. All P values were two-sided. All statistical analyses were performed with SPSS 23.0 (Inc, Chicago, IL).

| Baseline characteristics of patients
The median age of all the patients at baseline was 60.8 years (range, 37-85) with a male/female ratio of 2.18 ( Table 1). The frequency of organ involvement was kidney (79.2%), heart (68.3%), liver (13.9%), peripheral nerve (11.4%) and intestine (9.5%). Of the patients, 68.3% had more than one organ involved. The median value of NT-proBNP was 3885.4 pg/mL (range, 5-35000); the median value of troponin

| Expression frequency and organ tropism of three CT antigen genes in AL amyloidosis
As shown in Table 2 There was no significant correlation between organ propensity and MAGE-CT gene expression. Additionally, in each targeted organ subgroup, the rank of the positive expression rate in the newly diagnosed AL was MAGE-C1/CT7 > MAGE-C2/CT10 > MAGE A3, which was the same as in the overall cohort.

| Spectrum of MAGEC1/CT7 expression in different plasma cell dyscrasias
The expression of the MAGE-C1/CT7 gene was significantly highest in the plasma cell leukaemia and active myeloma groups and lowest in the MGUS group ( Figure 1). The level of MAGE-C1/CT7 gene expression in MM patients was higher than in the amyloidosis and MGUS groups (P < 0.001). Additionally, the level of MAGE-C1/ CT7 gene expression in the amyloidosis group was higher than in the MGUS group (P < 0.001).

| Correlation between CT antigens and prognostic factors in AL amyloidosis
The percentages of BM plasma cell infiltration positively correlated with the expression levels of MAGE-C2/CT10 (P = 0.008) and MAGE-A3 (P = 0.011). We did not find a correlation between MAGE-CT antigens and dFLC or NTproBNP. Additionally, there was no correlation between the β2-microglobulin (β2 MG), Lactate Dehydrogenase (LDH) 16

| Relationship between MAGE antigen and OS
After a median follow-up duration of 1266 days (range, 18-1863), the median survival was not reached for either group, and the 1and 2-year OS were 77.1% and 65.5% respectively. There was no significant difference in overall survival among the patients whose MAGE-CT antigens were positive or negative ( Figure 3).

| D ISCUSS I ON
Our results indicated that CT antigens were potentially effective molecular markers of AL amyloidosis and have clinical implications for monitoring treatment efficacy.
We found that CT antigens (CTA) were commonly expressed in AL amyloidosis. The most prevalent CTA was MAGE-C1/CT7,   The change in MAGE-CT gene expression paralleled the change in FLC, which was the same as that in myeloma. 7,18,20 In addition, one patient showed changes in MAGE before FLC changes. These results suggested that MAGE-C1/CT7 has the potential to detect disease relapse/progression at an earlier stage than the standard clinical monitoring method. These results also showed that the changes in MAGE and FLC could be a good supplement to the monitoring of  The prognostic role of the three CT antigens in AL amyloidosis was analysed. In addition to plasma cell (PC) percentage, we found that none of the MAGE CT genes correlated with β2MG, LDH or NT-proBNP, which are prognostic indictors for AL amyloidosis. The expression level of MAGE-C1/CT7 correlated with the level of cTnI.
Chromosomal abnormalities, including 1q21, t (11; 14), were associated with poor survival rates. 27 In this study, patients with MAGE A3 expression had more frequent 1q21 amplification. We did not find any correlation between MAGE CT gene expression and a propensity to specific affected organs (organ tropism). Organ tropism has been reported to be partially related to PC clones derived from particular IGLV genes. [28][29][30][31] Our observations in this study suggested that MAGE genes may not be potential factors regulating organ tropism. Because the overall survival for AL amyloidosis is very complex and mainly relies on the amount of amylogenic-free light chain and the degree of damage to the heart, we did not find any correlation between MAGE CT gene expression and overall survival. Due to the short follow-up time and limited cases, these conclusions need to be re-evaluated in larger sample sizes and during longer follow-up times.
Vaccine therapy is emerging in MM but not in AL amyloidosis. 23,[32][33][34] Low tumour burden and low tumour cell proliferative index are two advantages for immunotherapy in AL amyloidosis. 35 The specificity to malignant plasma cells and the common expression of these antigens in AL amyloidosis strongly suggest that they are promising targets for vaccine immunotherapy. Formulating vaccines with MAGE-CT antigens is an attractive strategy.
In conclusion, our study demonstrated that MAGE-CT genes, especially MAGE C1/CT7, were commonly expressed in AL amyloidosis, and the expression levels of MAGE CT genes could potentially be used as clinical indicators for auxiliary diagnoses and monitoring treatment efficacy in AL patients. However, these conclusions still need further verification through prospective studies with larger sample sizes.
Further investigation into the function of MAGE CT genes in AL amyloidosis is warranted to reveal novel therapeutic targets.

ACK N OWLED G EM ENTS
The study was supported by grants from the National Basic

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Jin Lu and Guo-Rui Ruan designed the study. Jin Lu performed data analysis. Guo-Rui Ruan did the RT-PCR examination. Yang Liu performed the data collection, and drafted the manuscript. Jin Lu, Xiaojun Huang and Kai-Yan Liu provided significant input on the manuscript and data analysis. Lei Wen, Ying Kang and Ling Ma helped to collect data and draft the manuscript. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.